Method for sequence determination using NMR

ABSTRACT

The invention relates to methods for analyzing polysaccharides. In particular, compositional and sequence information about the polysaccharides are derived. Some methods use NMR in conjunction with another experimental method, such as, capillary electrophoretic techniques for the analysis.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 from U.S.provisional application Ser. No. 60/381,940 filed May 20, 2002, theentire contents of which is incorporated by reference.

GOVERNMENT SUPPORT

Aspects of the invention may have been made using funding from theNational Institutes of Health Grant number GM57073 and CA090940.Accordingly, the Government may have rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods for analyzing compositional andsequence information of polysaccharides.

BACKGROUND OF THE INVENTION

Heparin and heparan sulfate glycosaminoglycans are complex acidicpolysaccharides that are involved in a variety of physiological andpathological conditions. Advances in different areas of biology haveelucidated the potential roles of HSGAGs in key biological processes(Casu and Lindahl, 2001; Lindahl, 2000; Sasisekharan and Venkataraman,2000; Shriver et al., 2002) including thrombosis (Petitou et al., 1999),angiogenesis (Sasisekharan et al., 1997), viral invasion (Chen et al.,1997; Fry et al., 1999; Shukla et al., 1999) and tumor growth (Hulett etal., 1999; Vlodavsky et al., 1999; Liu et al. 2002). The repeat unit ofa HSGAG polymer is a disaccharide comprising a uronic acid (U), whichcan exist in two different epimeric forms—α-L-iduronic (I) orβ-D-glucuronic (G), linked 1→4 to a α-D-glucosamine residue (A). Thereare variations within the disaccharide unit in the form of sulfation atthe 2-O position of the uronic acid, 3-O and 6-O position of theglucosamine and sulfation or acetylation of the N-position of theglucosamine (Casu and Lindahl, 2001).

Perhaps the best studied structure-activity relationships in HSGAGs is apentasaccharide sequence in heparin that specifically binds to andactivates antithrombin-III thereby playing an inhibitory role in theblood coagulation cascade (Bourin and Lindahl, 1993). Heparin and itsderivatives, low molecular weight heparins (LMWHs), are the most widelyused clinical agents for prevention of deep vein thrombosis aftersurgery (Breddin, 2000) and for prevention of myocardial infarctionafter coronary invasion procedures (Cohen, 1999). Based on theanticoagulant properties of heparin, new therapeutic applications ofheparin are being envisaged (Rosenberg, 2001). A synthetic version ofthe pentasaccharide has been used as an antithrombotic drug (Turpie etal, 2001).

In order to understand the structure-activity relationship of HSGAGs,several analytical tools have been developed for sequencingoligosaccharides which include gel electrophoresis (Turnbull et al.,1999), HPLC (Vives et al., 1999), matrix assisted laserdesorption/ionization mass spectrometry (MALDI-MS) (Venkataraman et al.,1999) and nanoelectrospray mass spectrometry (Pope et al., 2001). Theseanalytical tools have been applied to dissect the HSGAG oligosaccharideinto smaller fragments using a battery of depolymerizing enzymes andother chemical methods and determining the sequence of theoligosaccharides based on specific properties of the smaller fragments(Kreuger et al. 2001).

SUMMARY OF THE INVENTION

The invention relates, in part, to an analytical tool for analyzingoligosaccharides, such as HSGAGs. Determining the sequence and/orcomposition of an oligosaccharide is helpful for elucidating thestructure-function relationship of oligosaccharides in key biologicalprocesses.

In some aspects, a method of determining the composition of anoligosaccharide is provided. The method involves obtaining a measurementof a first property of the oligosaccharide using NMR spectroscopy, andobtaining a measurement of a second property of the oligosaccharide by asecond experimental method, wherein the first and second propertiesdetermine the composition. In one embodiment, the second property of theoligosaccharide is measured by capillary electrophoresis.

A method of analyzing an oligosaccharide is provided according to otheraspects. The method involves obtaining a measurement of a first type ofdisaccharide linkage of the oligosaccharide by a first experimentalmethod, and obtaining a measurement of a second type of disaccharidelinkage of the oligosaccharide by a second experimental method, toanalyze the oligosaccharide. In one embodiment the first type ofdisaccharide linkage is measured by NMR spectroscopy. In anotherembodiment the second type of disaccharide linkage is measured bycapillary electrophoresis.

According to another aspect, a method of analyzing an oligosaccharide,by identifying a first property of the oligosaccharide by NMRspectroscopy, and identifying a second property of the oligosaccharideby capillary electrophoresis, to analyze the oligosaccharide isprovided.

The methods, in some embodiments, involve determining possible sequencesof the oligosaccharide that are consistent with the measurement from theNMR spectroscopy and second experimental method.

In other embodiments the methods involve constructing a list of possiblesequences based on the measurement from the NMR spectroscopy, andeliminating sequences from the list of possible sequences that are notconsistent with the measurement of the second experimental method.

The second experimental method may be used to distinguish the reducingand non-reducing ends of the oligosaccharide or fragments thereof. Inone embodiment, the second experimental method includes chemicaldegradation. In another embodiment the second experimental methodincludes end-labeling.

The second experimental method may also be used to determine thesignature of the reducing end of the oligosaccharide or fragmentsthereof. In one embodiment the signature of the reducing end isdetermined with capillary electrophoresis. The second experimentalmethod may allow the determination of the sulfation pattern of thesecond type of disaccharide linkage.

The NMR spectroscopy, in some embodiments, includes the determination ofthe sulfation pattern of the oligosaccharide or fragments thereof. Inanother embodiment the NMR is performed to identify and quantify bothreducing and non-reducing ends.

The methods may optionally involve obtaining a measure of an additionalproperty of the oligosaccharide by a third experimental method tofurther eliminate sequences not consistent with measurements obtainedfrom the third experimental method.

In some embodiments the NMR spectroscopy is 1D proton or 2D COSY/TOCSY.In other embodiments, the NMR spectroscopy is HSQC, DQF-COSY, NOESY orROESY. The NMR spectroscopy may also be any combination of the above.

The NMR spectroscopy may be performed on the oligosaccharide in itsintact form. Alternatively, it may be performed on the oligosaccharidein a fragmented form. The fragmented form may be produced by enzymaticdigestion. Enzymatic digestions may be complete or, in the alternative,partial.

The second experimental method may also involve digesting theoligosaccharide to a fragmented form, e.g., optionally produced byenzymatic digestion. In one embodiment the enzymatic digestion iscomplete.

In another aspect, a method of generating a list of possible sequencesof an oligosaccharide is provided. The method involves defining a set ofproperties of the oligosaccharide by performing NMR spectroscopy and asecond experimental method, wherein the NMR spectroscopy provides ameasurement of a first type of disaccharide linkage and the secondexperimental method provides a measurement of a second type ofdisaccharide linkage, and constructing a list of possible sequencesbased on the set of properties of the oligosaccharide. In one embodimentthe NMR spectroscopy includes a measure of the monosaccharidecomposition of the oligosaccharide.

The method may involve a data structure which represents the propertiesas non-character values. In one embodiment the data structure includes avalue for each type of monosaccharide. In another embodiment the datastructure encodes a value for each type of disaccharide linkage. Inanother embodiment the values are binary.

A list of possible sequences of an oligosaccharide produced from themethods is also provided.

In the above aspects and embodiments the labels of “first” and “second”experimental methods are not intended to denote the order in which theexperiments need be performed. In some embodiments, the NMR spectroscopymay be performed before the other experimental methods, while in otherembodiments, the NMR spectroscopy may be performed after the othermethod. In still other embodiments the two or more experimental methodsmay be performed concurrently.

Each of the embodiments of the invention can encompass variousrecitations made herein. It is, therefore, anticipated that each of therecitations of the invention involving any one element or combinationsof elements can, optionally, be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the (A) disaccharide building block ofheparin and heparan sulfate polysaccharides (left) with positions ofsulfation marked as X (Y position can be sulfated or acetylated). Thehexadecimal code (middle) to represent the disaccharide repeat unit inthe PEN framework was described in our previous study (Venkataraman etal., 1999). The decomposition of the disaccharide hexadecimal code to abase₄ code for the uronic acid and base₈ (octal) code for theglucosamine is shown on in (B) The base₄ codes for the (i) uronic acids,(ii) octal codes for glucosamines, (iii) signed hexadecimal codes forA-U linkages and (iv) ΔU-A linkages observed in the oligosaccharidesused in this study.

FIG. 2 shows the (A) ¹H 500 MHz spectrum of synthetic pentasaccharideP1. (B) ¹H 500 MHz spectrum of synthetic pentasaccharide P2.Characteristic proton chemical shifts of the constituent monosaccharidesare marked accordingly.

FIG. 3 illustrates the ¹H 500 MHz spectra of H10 decasaccharide (A). Theline broadening is due to the presence of paramagnetic impurity;addition of deuterated EDTA provides a better resolved spectrum (B). Theanomeric region expansion with the signals assignment is shown in (C).

DETAILED DESCRIPTION

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) are cell surfacepolysaccharides that bind to a multitude of signaling molecules,enzymes, pathogens and modulate critical biological processes rangingfrom cell growth and development to anticoagulation and viral invasion.Heparin has been widely used as anticoagulant in a variety of clinicalapplications for several decades. The heterogeneity and complexity ofHSGAGs pose significant challenges to their purification andcharacterization of structure-function relationship.

Several analytical techniques including gel electrophoresis,chromatography and mass spectrometry have been successfully applied tosequence small amounts of HSGAG oligosaccharides. The prerequisite forthe success of most of these techniques is predictable and controlleddepolymerization of HSGAG oligosaccharide into smaller fragments using acombination of enzymatic and chemical degradation methods. Importantly,for some of the sequencing strategies, the use of multiple exo-enzymesare required to accurately determine the different modifications of thedisaccharide units.

A methodology was developed that utilizes experimental methods tocharacterize oligosaccharides. The invention, relates in some aspects toa method for analyzing an oligosaccharide, such as an HSGAG. Thesemethods, in some embodiments, encompass determining the presence oridentity of an oligosaccharide in a sample through the analysis of theoligosaccharide. In other embodiments, methods for assessing the purityof an oligosaccharide in a sample are provided. The term “analyzing” asused herein refers to the identification of one or more properties of anoligosaccharide. In some instances the analysis may be extensive andprovide considerable information about the composition of anoligosaccharide. The term analyzing can encompass sequencing anoligosaccharide or determining the composition of an oligosaccharide.The term “determine the composition” refers to the identification ofenough properties of an oligosaccharide that the oligosaccharide can bedistinguished from other oligosaccharides. When the informationregarding the properties is compiled using the numerical PEN frameworkderived by the instant inventors the sequence of an oligosaccharide,including order of building blocks and linkage information, can becompiled.

The analysis of the oligosaccharide may involve identification ofmonomeric properties and linkage information. The importance ofidentifying the types of linkages between monosaccharides anddisaccharides of oligosaccharides can be illustrated using HSGAGs as anexample. The repeat unit of a HSGAG polymer is a disaccharide comprisinga uronic acid (U), which can exist in two different epimericforms—α-L-iduronic (I) of β-D-glucuronic (G), linked 1→4 to aα-glucosamine residue (A). There are variations within the disaccharideunit in the form of sulfation or acetylation of the N-position of theglucosamine. Characteristic proton and ¹³C chemical shifts have beenidentified for commonly occurring monosaccharides and heparin and therelative abundance of these monosaccharides can be quantitativelydetermined by integrating the proton signals. In addition tocharacterization of the monosaccharides, the anomeric proton signals ofthe glucosamines can be resolved further to identify linkage informationregarding the neighboring uronic acid (A-U linkage) with a definedepimeric and sulfation state.

The composition of an oligosaccharide can be determined by the methodsdescribed herein using two or more experimental techniques to measureproperties of the oligosaccharide. One of the experimental techniques isnuclear magnetic resonance (NMR). By utilizing NMR the number ofexperimental constraints required to sequence HSGAG oligosaccharides canbe reduced. The NMR can be utilized with at least one other type ofexperimental technique. It has been demonstrated herein that theintegration of NMR and capillary electrophoresis (CE) data sets with thehelp of the PEN framework can reduce the need for NOESY/ROESYexperiments which may require a lot more sample and more sophisticatedinstruments for reasonable sensitivity.

Thus, methods are provided to analyze the oligosaccharides to determinethe sequence of the oligosaccharide. Using a numerical PEN framework,the information derived from the compositional analysis, e.g., twodistinct linkage data sets, can be compiled to construct the sequence ina rapid, systematic and unbiased fashion. The numerical nature of thePEN framework facilitates moving between monosaccharide, disaccharideU-A and A-U linkage information using simple mathematical operationsthus facilitating a systematic and unbiased way of rapidly arriving at aHSGAG sequence.

The examples discussed below demonstrate the use of the methodology andthe computational approach described herein. Examples 1 and 2 describepentasaccharides containing both I_(2S) and G. These examplesdemonstrate the value of determining the signature of the reducing end(methylation) for application of the analytical methods describedherein. Once all of the A-U and U-A linkages are determined, theknowledge of the reducing or non-reducing end enables the determinationof the sequence by moving forward or backward. Decasaccharide H10 is oneof the most complex decasaccharides that has been characterized andverified using a combination of analytical tools.

Example 3 highlights the strengths of the methods described herein incomparison with other analytical tools that have been used forcharacterization of H10 in the past. Earlier sequencing approaches forthis decasaccharide required numerous steps. Using the methods describedherein we arrived at the sequence of H10 in an unbiased fashion byobtaining two distinct sets of A-U and U-A linkage information that werequantitatively determined using a minimal set of experimental data. Thisexample illustrates the flexibility of the computational method toconstruct a list of all possible combinations of sequences satisfyingthe linkage and monosaccharide composition obtained from NMR data andelimination of sequences that did not satisfy the CE data.

Thus, the list of possible sequences can be constructed based on theidentified properties, such as the charge, the nature and number ofunits of the oligosaccharide, the nature and number of chemicalsubstituents on the units, disaccharide linkage, reducing andnon-reducing ends, and the stereospecificity of the oligosaccharide,which reveal information about the building blocks of theoligosaccharide determined from the measurements of an experimentalmethod.

The structural properties of oligosaccharides may provide usefulinformation about the function of the oligosaccharide. For instance, theproperties of the oligosaccharide may reveal the entire sequence ofunits of the oligosaccharide, which is useful for identifying theoligosaccharide. Similarly, if the sequence of the oligosaccharide waspreviously unknown, the structural properties of the oligosaccharide areuseful for comparing the oligosaccharide to known oligosaccharideshaving known functions. The properties of the oligosaccharide may alsoreveal that a oligosaccharide has a net charge or has regions which arecharged. This information is useful for identifying compounds that theoligosaccharide may interact with or predicting which regions of aoligosaccharide may be involved in a binding interaction or have aspecific function.

The invention is useful for identifying properties of oligosaccharides.A “property” as used herein is a characteristic (e.g., structuralcharacteristic) of the oligosaccharide that provides information (e.g.,structural information) about the oligosaccharide. A compilation ofseveral properties of a oligosaccharide may provide sufficientinformation to identify a chemical unit or even the entireoligosaccharide but the property of the oligosaccharide itself does notencompass the chemical basis of the chemical unit or oligosaccharide.Due to the complexity of the oligosaccharide, a property may identify atype of monomeric building block of the oligosaccharide. The units ofthe oligosaccharides have more variables in addition to its basicchemical structure. For example, the oligosaccharide may be acetylatedor sulfated at several sites on the building block, or it may be chargedor uncharged. Thus, one property of an oligosaccharide may be theidentity of one or more basic building blocks of the oligosaccharides.

A basic building block alone, however, may not provide information aboutthe charge and the nature of substituents of the saccharide ordisaccharide. For example, a building block of uronic acid may beiduronic or glucuronic acid. Each of these building blocks may haveadditional substituents that add complexity to the structure of thebuilding block. A single property, however, may not identify suchadditional substitutes charges, etc., in addition to identifying acomplete building block of a oligosaccharide. This information, however,may be assembled from several properties. Thus, a property of anoligosaccharide as used herein encompasses a monosaccharide ordisaccharide building block of an oligosaccharide. The NMR methodsdescribed herein are useful for identifying information about basicmonosaccharide building blocks.

The type of property that will provide structural information about theoligosaccharide is a property such as charge, molecular weight, natureand degree of sulfation or acetylation, or type of saccharide.Properties include but are not limited to charge, chirality, nature ofsubstituents, quantity of substituents, molecular weight, molecularlength, compositional ratios of substituents or units, type of basicbuilding block of a oligosaccharide, hydrophobicity, enzymaticsensitivity, hydrophilicity, secondary structure and conformation (i.e.,position of helices), spatial distribution of substituents, ratio of oneset of modifications to another set of modifications (i.e., relativeamounts of 2-O sulfation to N-sulfation or ratio of iduronic acid toglucuronic acid), binding sites for proteins, and linkage information.Other properties will easily be identified by those of ordinary skill inthe art. A substituent, as used herein is an atom or group of atoms thatsubstitute a unit, but are not themselves the units.

It has been discovered that the use of NMR can significantly reduce thenumber of experimental constraints required to derive enough propertyinformation to identify a complete sequence of an oligosaccharide. Byproviding information regarding specific linkages and monosaccharidecompositions NMR dramatically improves sequencing techniques.

Thus, one embodiment of the methods utilizes the strengths of NMR tomeasure A-U linkages and an orthogonal set of U-A linkage informationobtained from CE′ to construct the sequence of an oligosaccharide. NMRis a powerful tool that can be used to determine numerous parametersdefining the sequence of an intact oligosaccharide includingmonosaccharide composition, sulfation pattern and linkage betweenglucosamine and uronic acid (A-U). These parameters can be readilydetermined independent of sequence length and variability of buildingblocks, using a single series of simple 1D proton and 2D COSY/TOCSYexperiments. Thus, by combining the distinct linkage information betweenadjacent monosaccharides obtained from NMR (A-U linkage information)with the U-A linkage information obtained from a single capillaryelectrophoresis experiment to rapidly arrive at the sequence of HSGAGoligosaccharides.

Interpretation of NMR spectra of HSGAGs, in the past, has had certainlimitations due to overlaps in proton signals and absence of measurablecoupling constants. Also the sensitivity of this technique is lower thanthose based on detection of chromatographic effluents (Turnbull et al,1999; Vives et al, 1999; Kreuger et al, 2001) and on mass spectrometry(Pope et al., 2001; Venkataraman et al., 1999). Thus, it was verydifficult to characterize samples that are only available in smallquantities. It has been discovered that despite these limitations NMRcan provide powerful analytical information useful in sequencing.

NMR spectroscopy is an analytical tool that allows for the determinationof molecular structure. Utilizing the magnetic properties of somenuclei, the nuclear spins of the nuclei can be oriented randomly with anexternal magnetic field. Oriented nuclei that are subsequentlyirradiated at the correct frequency will absorb energy and transition toa higher energy state. Upon relaxation this energy is emitted anddetected in various NMR systems. This irradiation of the nuclei occur inpulses. In basic one dimensional (1D) NMR the excitation is producedfrom a single pulse and emitted radiation is detected as free inductiondecay (FID). In two dimensional (2D) NMR spectroscopy the nuclei isirradiated with two pulses, and acquisition of the FID occurs at manytime points with a delay between the pulses.

There are many types of 2D spectroscopy which include: COSY, TOSCY,NOESY and ROESY. COSY (Correlated Spectroscopy) is helpful indetermining the energy that is arising from neighboring protons. This ishelpful if there is overlap or second order coupling. Spin-spin couplingin COSY allows the spectrum to yield through bond interactions. Another2D NMR technique capable of measuring through bond interactions is TOSCY(Total Correlated Spectroscopy) which identifies the protons thatproduce signals within a spin system. COSY and TOSCY can be combined fora more powerful structural analysis.

Other 2D NMR techniques allows the measurement of through spaceinteractions. These methods are referred to as NOESY (Nuclear OverhauserEffect Spectroscopy) and ROESY (Rotational Nuclear Overhauser EffectSpectroscopy). NOESY identifies the signals emitted from protons thatare close in space by not directly connected by bonding. The NOESYspectra give through space correlations as the change in intensity ofmultiplets from neighboring nuclei upon irradiation can be extensivelymeasured. In instances where NOESY signals are weak, ROESY can be used,a similar technique that has cross peaks that are only negative. Othertechniques may be used and would be apparent to those of ordinary skillin the art.

The NMR spectroscopy useful in the methods described herein may be 1Dproton or 2D COSY/TOCSY in some embodiments. 1D proton NMR and 2DCOSY/TOCSY NMR spectra provide quantitative information on multipleparameters including monosaccharide composition, sulfation states andA-U linkage information that define the sequence of an oligosaccharide.Furthermore, NMR provides an accurate method for direct quantificationof the iduronic and glucuronic acid content in a sequence.

In the method of capillary gel-electrophoresis, reaction samples may beanalyzed by small-diameter, gel-filled capillaries. The small diameterof the capillaries (50 μm) allows for efficient dissipation of heatgenerated during electrophoresis. Thus, high field strengths can be usedwithout excessive Joule heating (400 V/m), lowering the separation timeto about 20 minutes per reaction run, therefor increasing resolutionover conventional gel electrophoresis. Additionally, many capillariesmay be analyzed in parallel, allowing amplification of generatedoligosaccharide information.

Currently, saccharide fragments are detected in capillaryelectrophoresis by monitoring at 232 nm, the wavelength at which theΔ^(4,5) double bond, generated upon heparinase cleavage, absorbs.However, other detection methods are possible. First, nitrous acidcleavage of heparin fragments, followed by reduction with ³H-sodiumborohydride yields degraded fragments having a ³H radioactive tag. Thisrepresents both a tag which may be followed by capillary electrophoresis(counting radioactivity) or mass spectrometry (by the increase in mass).Another method of using radioactivity would be to label the heparinfragment with S³⁵. Similar to the types of detection possible for³H-labeled fragments, S³⁵ labeled fragments may be useful forradioactive detection (CE) or measurement of mass differences (MS).

Especially in the case of S³⁵, this detection will be powerful. In thiscase, the human sulfotransferases may be used to label specifically acertain residue. This will give additional structural information.

Nitrous acid degraded fragments, unlike heparinase-derived fragments, donot have a UV-absorbing chromophore. For CE, two methods may be used tomonitor fragments that lack a suitable chromophore. First is indirectdetection of fragments. We may detect heparin fragments with our CEmethodology using a suitable background absorber, e.g.,1,5-napthalenedisulfonic acid. The second method for detection involveschelation of metal ions by saccharides. The saccharide-metal complexesmay be detected using UV-Vis just like monitoring the unsaturated doublebond.

For determining the reducing or non-reducing ends a variety ofexperimental methods may be used including chemical degradation,end-labeling and capillary electrophoresis. In some embodiments, theends may be determined by measuring a signature of a reducing ornon-reducing end. For some oligosaccharides, the signature of thereducing end is methylation, and this signature may also be determinedby capillary electrophoresis as well as other methods known in the art.

The reducing end of an oligosaccharide may be distinguished from thenon-reducing end using mass tags, for instance. All of these tagsinvolve selective chemistry with the anomeric OH (present at thereducing end of the oligosaccharide), thus labeling occurs at thereducing end of the chain. One common tag is 2-aminobenzoic acid whichis fluorescent. In general tags involve chemistry of the followingtypes: (1) reaction of amines with the anomeric position to form imines(i.e., 2-aminobenzoic acid), hydrazine reaction to form hydrazones, andreaction of semicarbazones with the anomeric OH to form semicarbazides.Commonly used tags (other than 2-aminobenzoic acid) include thefollowing compounds:

-   1. semicarbazide-   2. Girard's P reagent-   3. Girard's T reagent-   4. p-aminobenzoic ethyl ester-   5. biotin-x-hydrazide-   6. 2-aminobenzamide-   7. 2-aminopyridine-   8. anthranilic acid-   9. 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein-   10. 8-aminonaphthalene-1,3,6-trisulfonic acid-   11. 2-aminoacridone

The properties, in some aspects of the invention, may be determined forthe oligosaccharide in its intact or a fragmented form. Fragments ofoligosaccharides in some embodiments of the invention can be produced byenzymatic digestion. In some embodiments the digestion is complete, orit may be a partial digestion.

Oligosaccharide fragments may be degraded using enzymes such as heparinlyase enzymes or nitrous acid and they may also be modified usingdifferent enzymes that transfer sulfate groups to the positionsmentioned earlier or remove the sulfate groups from those positions. Themodifying enzymes are exolytic and non-processive which means that theyjust act once on the non-reducing end and will let go of the heparinchain without sequentially modifying the rest of the chain. For each ofthe modifiable positions in the disaccharide unit there exits amodifying enzyme. An enzyme that adds a sulfate group is called asulfotransferase, and an enzyme that removes a sulfate group is called asulfatase. The modifying enzymes include 2-O sulfatase/sulfotransferase,3-O sulfatase/sulfotransferase, 6-O sulfatase/sulfotransferase andN-deacetylase-N-sulfotransferase. The function of these enzymes isevident from their names, for example a 2-O sulfotransferase transfers asulfate group to the 2-O position of an iduronic acid (2-O sulfatedglucuronic acid is a rare occurrence in the HSGAG chains) and a 2-Osulfatase removes the sulfate group from the 2-O position of an iduronicacid.

HSGAG degrading enzymes include heparinase-I, heparinase-II,heparinase-III, D-glucuronidase and L-iduronidase. The heparinasescleave at the glycosidic linkage before a uronic acid. Heparinase Iclips at a glycosidic linkage before a 2-O sulfated iduronic acid.Heparinase-III cleaves at a glycosidic linkage before an unsulfatedglucuronic acid. Heparinase-II cleaves at both Hep-I and Hep-IIIcleavable sites. After cleavage by the heparinases the uronic acidbefore which the cleavage occurs loses the information of iduronic vs.glucuronic acid because a double bond is created between the C4 and C5atoms of the uronic acid.

Glucuronidase and iduronidase, as their name suggests cleave at theglycosidic linkage after a glucuronic acid and iduronic acidrespectively. Nitrous acid clips randomly at glycosidic linkages after aN-sulfated hexosamine and converts the six membered hexosamine ring to a5 membered anhydromannitol ring.

As used herein, the term “oligosaccharide” is used interchangeably withthe term “polysaccharide”. An “oligosaccharide” is a biopolymercomprised of linked saccharide or sugar units. As used herein withrespect to linked units of a oligosaccharide, “linked” or “linkage”means two entities are bound to one another by any physicochemicalmeans. Any linkage known to those of ordinary skill in the art, covalentor non-covalent, is embraced. Such linkages are well known to those ofordinary skill in the art. Natural linkages, which are those ordinarilyfound in nature connecting the chemical units of a particularoligosaccharide, are most common. Natural linkages include, forinstance, amide, ester and thioester linkages. The units of anoligosaccharide analyzed by the methods of the invention may be linked,however, by synthetic or modified linkages. Oligosaccharides where theunits are linked by covalent bonds will be most common but also includehydrogen bonded, etc.

The oligosaccharide is made up of a plurality of chemical units. A“chemical unit” as used herein is a building block or monomer which maybe linked directly or indirectly to other building blocks or monomers toform an oligosaccharide. The oligosaccharide preferably is aoligosaccharide of at least two different linked units. Anoligosaccharide is a biopolymer composed of monosaccharides linked toone another. In many oligosaccharides the basic building block of theoligosaccharide is actually a disaccharide unit which may be repeatingor non-repeating. Thus, a unit when used with respect to aoligosaccharide refers to a basic building block of an oligosaccharideand may include a monomeric building block (monosaccharide) or a dimericbuilding block (disaccharide).

A “plurality of chemical units” is at least two units linked to oneanother. The oligosaccharides may be native or naturally-occurringoligosaccharides which occur in nature or non-naturally occurringoligosaccharides which do not exist in nature. The oligosaccharidestypically include at least a portion of a naturally occurringoligosaccharide. The oligosaccharides may be isolated or synthesized denovo. For example, the oligosaccharides may be isolated from naturalsources e.g. purified, as by cleavage and gel separation or may besynthesized e.g., by chemical synthesis.

A data structure for representing the properties of the oligosaccharideis also provided. In some embodiments the data structure represents theproperties as non-character values. These values in some embodiments canbe a binary value. In some embodiments the building blocks are the typesof monosaccharides and disaccharide linkages of the oligosaccharide.

The rapid sequencing methodology for polysaccharides using chemical andenzymatic tools followed by numerical analysis techniques is describedin detail in Venkataraman, G., et al., Science, 286, 537–542 (1999), andU.S. patent application Ser. Nos. 09/557,997 and 09/558,137, both filedon Apr. 24, 2000 and having common inventorship, all of which arespecifically incorporated by reference.

EXAMPLES

Materials and Methods

The synthetic pentasaccharides P1 and P2 corresponding to the activesequence of heparin for AT-III binding were a gift from M. Petitou,Sanofi-Synthelabo, Toulouse, France. The decasaccharide H10, kindlyprovided by R. J. Linhardt (University of Iowa), was obtained byfractionation of heparinase digest of pig mucosal heparin, on an AT-IIIcolumn as described earlier (Toida et al., 1996)

NMR Spectroscopy:

The oligosaccharide samples were prepared by dissolving 2 mg of thepentasaccharide and 150 μg of H10 in 0.5 ml of D₂O 99.99%. Due to signalbroadening caused by paramagnetic ions in H10, deuterated EDTA was addedto the sample to remove these ions. (Neville et al., 1989) The ¹H-NMRspectra were recorded at 500 MHz on a Bruker AMX 500 spectrometer at 60°C. with presaturation of the residual water signals and with recycledelay of 12 seconds; a 45° pulse was used. 2D DQF-COSY (Double QuantumFiltered-COSY) and TOCSY were measured in phase-sensitive mode usingTPPI (Time Proportional Phase Incrementation), and a shifted squaresine-bell function was applied before Fourier transformation. 32 and 512scans for each FID were used for the pentasaccharide and thedecasaccharide, respectively.

Compositional Analysis using Capillary Electrophoresis (CE):

Compositional analysis of the oligosaccharides was completed byexhaustive enzymatic digest of a 30 μM sample followed by capillaryelectrophoresis (CE) as described earlier (Venkataraman et al;, 1999).Briefly, to 1 nmol of oligosaccharide was added 200 nM of heparinases I,II, and III in 25 mM sodium acetate, 100 mM NaCl, 5 mM calcium acetatebuffer, pH 7.0. The reaction was allowed to proceed at 30° C. overnightand then analyzed by CE in reverse polarity with a running buffer of 50mM Tris/phosphate/10 μM dextran sulfate, pH 2.5.

Incorporating CE and NMR Data as Constraints using PEN Framework:

The PEN is a numerical notation scheme that encodes the sulfationpattern of a disaccharide building block as a series of binary on/offstates and epimerization of the uronic acid as a + or − sign bit leadingto a signed hexadecimal coding scheme (Venkataraman et al., 1999).Although, the PEN framework was originally developed to encode a U-Adisaccharide building block (Di), it was mathematically decomposed intoa base₄ code for representing the uronic acid monosaccharide (U) and abase₈ code for representing the glucosamine monosaccharide (A) (FIG. 1).Note that for the ΔU-A linkages the * used in the G/I position indicatesthat the epimeric state of the uronic acid is undetermined. It is alsoimportant to note that the signed hexadecimal codes representing the A-Ulinkages involves rearrangement of the 3 binary digits encoding A andtwo binary digits encoding U from the original PEN framework. As aresult of the rearrangement, the + and − sign is used to represent 6-Osulfation (where + represents unsulfated and − represents sulfated) ofthe glucosamine instead of the epimeric state of the uronic acid sincethe 6-O sulfation is in the left most position of the A-U disaccharidecode. Therefore there is no “extra” binary digit that has been added forrepresenting the A-U disaccharide and we still use the signed base₁₆hexadecimal code. Further the PEN framework was also used to encode anA-U disaccharide unit (Di′) by transposing the 3 bits that encode forthe sulfation state of the glucosamine with the 2 bits that encode theepimeric and sulfation state of the uronic acid (Table 1). Theinformation obtained from NMR and CE data is shown in A. Columns 1–3indicate the number of linkages between glucosamine residues (colored ingray) the uronic acids in column 4 obtained from NMR data. Columns 5–7indicate the linkages between uronic acid and the glucosamines obtainedfrom CE data. The sequences that satisfy the monosaccharide compositionand A-U linkage information (Di′) are shown in B. Application of the U-Alinkages from CE data reduces L_(NMR) to the final correct sequence.

TABLE 1 Sequence assignment of H10

1-D proton NMR spectrum along with the 2D COSY, HSQC (HeteronuclearSingle Quantum Coherence) and TOCSY spectra provide data on the chemicalshifts and coupling constants of most the ring protons of theconstituent monosaccharides. This data was used to uniquely identify themonosaccharides (Ui and Ai) and obtain the number of monosaccharides fora given length of the sequence. In addition to the identity of theglucosamine monosaccharides, their characteristic anomeric chemicalshifts were further resolved to identify their linkages to adjacenturonic acids (Ai-Ui linkages defining Di′ disaccharides). The Ui, Ai andDi′ information was used to build a list of all the possible sequencessatisfying this data (L_(NMR)). The sequences in L_(NMR) represent acomprehensive sample space without any bias towards commonly occurringsequences.

Disaccharide compositional analysis using CE provides accurateinformation on the sulfation pattern of a ΔU-A disaccharide, thusidentifying all the U-A linkages (±Di) whose sign bit is not known dueto the Δ⁴⁻⁵ unsaturated bond. Incorporating the disaccharide linkages±Di obtained from CE data eliminates most of the sequences from L_(NMR)converging on a single sequence.

Results

Example 1 Pentasaccharide P1

Several characteristic chemical shifts of the monosaccharide anomericprotons were observed in the 1D proton NMR spectrum of thepentasaccharide (FIG. 2A). The 1D proton signals along with the 2D COSYand TOCSY spectra were used to assign the monosaccharides. The signalpatterns at 5.648, 5.438 and 5.041 ppm were assigned to the anomericprotons of N-sulfated glucosamines (A_(NS,6X)). Further, the 6-Osulfation of all these glucosamines were confirmed by TOCSY. The signalat 3.43/57.5 ppm indicates the presence of an O-methyl group linked tothe reducing terminal. In addition, the presence of the methyl group atthe reducing end also accounts for the absence of the typical reducingend carbon chemical shift (92–93 ppm). The chemical shifts at 5.251 and4.635 are in agreement with an I_(2S) and G respectively. The anomericproton signals at 5.648 and 5.438 are distinguished further as arisingfrom A_(NS,6S)-I_(2S) and A_(NS,6S)-G respectively.

Integration of these peaks (Guerrini et al., 2001) gave the relativemolar abundance of the glucosamines asA_(NS,6S)-I_(2S):A_(NS,6S)-G:A_(NS,6S)=1:1:1 and that of the uronicacids as I_(2S):G=1:1. Thus from the 1D and 2D NMR data the identityA_(i)=[5₈; 5₈; 5₈ (A_(NS,6S))], U_(i)=[1₄ (I_(2S)); 2₄ (G)] and relativeabundance (m5=3, n1=n2=1) of the monosaccharides constituting thesequence were determined. Further the linkages Di′=[-5₁₆(A_(NS,6S)-I_(2S)); -6₁₆ (A_(NS,6S)-G)] were also obtained form NMRdata. Based on this information from the NMR data there can be twopossible (L_(NMR)) pentasaccharide sequences: 5₈ 1₄ 5₈ 2₄ 5₈(A_(NS,6S)-1_(2S)-A_(NS,6S)-G-A_(NS,6S,OMe)) and 5₈ 2₄ 5₈ 1₄ 5₈(A_(NS,6S)-G-A_(NS,6S)-I_(2S)-A_(NS,6S,OMe)).

Capillary electrophoresis of the fragments formed by complete digestionof the pentasaccharide with the heparinases resulted in two peakscorresponding to a trisulfated ΔU_(2S)A_(NS,6S) and a disulfateddisaccharide ΔUA_(NS,6S) thus defining ±Di=[±D₁₆; ±5₁₆]. The relativemolar abundance of these two disaccharides was calculated as 1:1 byintegration of the CE signals and normalizing the peak areas using aninternal calibration. The migration time of the ±D₁₆ disaccharide wasslightly different from the standard indicating that the methylatedglucosamine is a part of the trisulfated ±D₁₆ disaccharide. Thus thedata from CE fixes the sulfation state of methylated reducing enddisaccharide. Incorporating the constraints from CE data eliminated oneof the sequences from L_(NMR) thus converging on 5₈ 2₄ 5₈ 1₄ 5₈(A_(NS,6S)-G-A_(NS,6S)-I_(2S)-A_(NS,6S,OMe)).

Example 2 Pentasaccharide (P2)

From the 1D proton spectrum (FIG. 2B) the signal pattern (anomeric peaksat 5.64/99.6 ppm, 5.50/98.1 ppm) is consistent with N-sulfated,6-O-sulfated glucosamines (A_(NS,6S)), and A* bearing an extra 3-Osulfate group. Similar to Example 1, the signal pattern at 3.43/57.5 ppmcorresponds to a glucosamine with a methylated reducing end. Also theanomeric chemical shift at 5.64 ppm arises from a A_(NS,6S) linked to Gas shown in Example 1. Signals at 5.20/101.7 ppm and 4.78/72.4 ppm agreewith H1 and H5 of I_(2S) residue, and the anomeric signal at 4.6/103.3ppm with G (Mulloy and Johnson, 1987; Yates et al;, 1996). Thus Ui=[1₄(I_(2S)); 2₄ (G)] and Ai=[5₈; 5₈ (A_(NS,6S)); 7₈ (A*)]. In this case wehave only one of the two elements of Di′ defined=[-6₁₆ (A_(NS,6S)-G)].Incorporating the inferences from NMR data as constraints we getL_(NMR)=2 sequences: 5₈ 2₄ 7₈ 1₄ 5₈(A_(NS,6S)-G-A*-I_(2S)-A_(NS,6S,OMe)) and 7₈ 1₄ 5₈ 2₄ 5₈ (A*-I_(2S-A)_(NS,6S-G-A) _(NS,6S,OMe))

The disaccharide composition analysis using CE resulted in a single peakcorresponding to a trisulfated disaccharide with a shifted migrationtime indicating the presence of the methylated glucosamine (±Di=[±D₁₆]).Using the data from CE one of the sequences from L_(NMR) was eliminatedto give the right sequence 5₈ 2₄ 7₈ 1₄ 5₈(A_(NS,6S)-G-A*-I_(2S)-A_(NS,6S,OMe)). This sequence is consistent withthe notion that the A_(NS,6S)-G linkage is resistant to cleavage byheparinase I, II and III due to the presence of the 3-O sulfated A*(Shriver, Z et al. 2000) thus resulting only in a single disaccharideobserved using CE.

Example 3 Decasaccharide (H10)

While the first two synthetic pentasaccharide examples clearly outlinethe methodology of our approach, the method is better illustrated by thelonger and more complex heparin derived oligosaccharide (H10). Thissequence is presently among the most complex heparin derivedoligosaccharide sequenced to date. It is important to point out thatmuch effort has gone into the isolation and sequencing of H10 (Toida etal., 1996). Due to its complexity, there were inaccuracies in itsstructure determination in the past and only recently using acombination of analytical tools, this sequence was established(Venkataraman et al., 1999; Shriver et al., 2000). NMR spectroscopy hasbeen used in the past to corroborate its sequence (Shriver et al., 2000)but only the monosaccharide composition was established and there wasbias in the interpretation of the NMR data based on the determinedsequence. Using these examples we highlight the flexibility of ourapproach in providing an unbiased assignment of complex heparin derivedoligosaccharide structures.

The signal line broadening of the proton spectrum of H10 (FIG. 3A) iscaused by the complexation of paramagnetic ions with the negativelycharged groups. Addition of EDTA provides a better resolved spectrum(FIG. 33B) by removal of these paramagnetic ions. (Neville, et al.,1989)

The assignment of the anomeric signals (FIG. 3C) and their respectiveproton patterns (Table 2), were carried out by COSY and TOCSYexperiments.

Table 2: ¹H Chemical Shifts of the Constituent Monosaccharides of theH10 Sample.

Chemical shifts are given in ppm downfield from trimethylsilylpropionate (TSP) as standard.

ΔU A_(NS,6S) ^(a) I_(2S) ^(a) A_(NS) I A_(NAc) G A* H1 5.521 5.42 5.205.369 5.019 5.386 4.668 5.460 H2 4.635 3.31 4.34 3.275 3.767 3.940 3.4153.483 H3 4.314 3.65 4.22 3.65 4.126 3.77 3.71 4.571 H4 6.0013.77(3.821)^(b) 4.13 3.77 4.102 3.75 3.804 4.040 H5 4.01 4.87 4.01 4.8294.01 3.71 4.261 H6 4.40 4.40 4.35 nd ^(a)two monosaccharide residues^(b)H4 chemical shift of the A_(NS,6S) residue following the nonreducing ΔU unit.

The signals detected between 4.2–4.4 ppm are in agreement with H-6proton from 6-O sulfated glucosamine. However since this chemical shiftlies in the crowded area of the spectrum and due to the presence ofminor impurities in the sample it was not possible to accuratelydetermine the molar abundance of glucosamines containing the 6-O sulfategroups. However disaccharide compositional analysis of H10 using CEindicated the presence of 3 major disaccharidecomponents—ΔU_(2S)-A_(NS,6S), ΔU-A_(NAc, 6S), ΔU-A* in the ratio 3:1:1,respectively, giving ±Di=[±D; ±4; ±7]. Thus the data from CE fixed the6-O sulfation of all the glucosamines.

The relative abundance of the glucosamine monosaccharides calculated bysignal integration were 5₈ (A_(NS,6S)): 7₈ (A*): 4₈ (A_(NAc,6S))=3:1:1,thus Ai=[5₈; 5₈; 5₈; 7₈; 4₈]. The two α anomeric signals at 5.20 and5.019 ppm arise from 2-O sulfated and non sulfated iduronic acid,respectively, as demonstrated by the chemical shift pattern. The only βproton signal of the spectrum (at 4.669 ppm) belongs to a glucuronicacid residue. Protons at 6 ppm and 5.521 ppm belong to the H4 and H1 ofthe ΔU residue. The H2 at 4.635 ppm indicates that the unsaturateduronic acid residue is 2-O-sulfated. The relative abundance of theuronic acid monosaccharides was calculated as I_(2S): I: G: ΔU_(2S) wereidentified in the ratio 2:1:1:1 respectively thereby defining Ui=[1₄;1₄; 0₄; 2₄; (*1)₄] (where * stands for ΔU since this bit is notdefined).

The chemical shift of the signal at 5.369 ppm agrees with a A_(NS,6S)linked to I. The ¹H anomeric chemical shift of a A_(NAc, 6S) is distinctfor A_(NAc,6S)-I (5.14–5.18 ppm) and A_(NAc,6S)-G (5.30–5.36 ppm)linkages (Cohen, 1999; Chuang, et al., 2001). The anomeric proton ofA_(NAC,6S) at 5.386 ppm confirms the presence of A_(NAc,6X)-G linkage inthe sequence. The chemical shift at 5.42 agrees with both A_(NS,6S)linked to I_(2S) and A_(NS,6S) at the reducing end. Since the ΔU residueis linked to an A_(NS,6S) unit and a second A_(NS,6S) is linked to I,two possibilities are left for the reducing end, one with A_(NS,6S) andthe other with A*. However, the chemical shift pattern associated withA* (H1, 5.464 ppm; H2, 3.480 ppm; H3, 4.564 ppm, H4, 4.041 ppm) is thesame as found by Yamada et al (1993) for a heparin tetrasaccharide withthis residue at the reducing end (chemical shifts in the Yamada et alpaper are systematically shifted about −0.03 ppm with respect to ourvalues). Thus the signal pattern of A* is consistent with its locationat the reducing end. Based on the relative abundance of these signalsall the elements of Di′ were defined as [-5₁₆; -5₁₆; -4₁₆; -2₁₆].Translating the Ai, Ui and Di′ to constraints using the PEN frameworkL_(NMR)=12 sequences were obtained (Table 1).

Eliminating the sequences from L_(NMR) that do not contain disaccharidelinkages corresponding to ±1Di resulted in a single sequence DDD4-7which is consistent with the H10 sequence obtained earlier (Shriver, etal., 2001).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, patents and patent publications that are recited in thisapplication are herein incorporated by reference.

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1. A method of generating a list of possible sequences of anoligosaccharide, comprising: defining a set of properties of theoligosaccharide by performing NMR spectroscopy and a second experimentalmethod, wherein the NMR spectroscopy provides a measurement of a firsttype of disaccharide linkage and the second experimental method providesa measurement of a second type of disaccharide linkage, and constructinga list of possible oligosaccharide sequences based on the set ofproperties of the oligosaccharide, wherein the oligosaccharide is aheparin/heparan sulfate (HSGAG) oligosaccharide.
 2. The method of claim1, wherein the NMR spectroscopy includes a measure of the monosaccharidecomposition of the oligosaccharide.
 3. The method of claim 1, furthercomprising a data structure which represents the properties asnon-character values.
 4. The method of claim 3, wherein the datastructure includes a value for each type of monosaccharide.
 5. Themethod of claim 3, wherein the data structure encodes a value for eachtype of disaccharide linkage.
 6. The method of claim 3, wherein thevalues are binary.
 7. The method of claim 1, wherein the NMRspectroscopy includes the determination of the sulfation pattern of theoligosaccharide or fragments thereof.
 8. The method of claim 1, whereinthe NMR spectroscopy is COSY, TOCSY, HSQC, DQF-COSY, NOESY, ROESY or acombination thereof.
 9. The method of claim 1, wherein the NMRspectroscopy is 1D proton or 2D COSY/TOCSY.
 10. The method of claim 1,wherein the NMR spectroscopy is performed on the oligosaccharide in afragmented form.
 11. The method of claim 10, wherein the fragmented formis produced by enzymatic digestion.
 12. The method of claim 11, whereinthe enzymatic digestion is with a heparinase, glucuronidase, iduronidaseor sulfatase.
 13. The method of claim 11, wherein the enzymaticdigestion is complete.
 14. The method of claim 1, wherein the NMRspectroscopy is performed on the oligosaccharide in its intact form. 15.The method of claim 1, wherein the second type of disaccharide linkageis measured by capillary electrophoresis.
 16. The method of claim 1,wherein the second experimental method includes chemical degradation.17. The method of claim 1, wherein the second experimental methodincludes end-labeling.
 18. The method of claim 1, wherein the secondexperimental method distinguishes the reducing and non-reducing ends ofthe oligosaccharide or fragments thereof.
 19. The method of claim 1,wherein the second experimental method includes digesting theoligosaccharide to a fragmented form.
 20. The method of claim 19,wherein the fragmented form is produced by enzymatic digestion.
 21. Themethod of claim 20, wherein the enzymatic digestion is with aheparinase, glucuronidase, iduronidase or sulfatase.
 22. The method ofclaim 20, wherein the enzymatic digestion is complete.